Erythromycin for myotonic dystrophy type 1: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial.

Background: Myotonic dystrophy type 1 (DM1) is a devastating multisystemic disorder caused by a CTG repeat expansion in the DMPK gene, which subsequently triggers toxic RNA expression and dysregulated splicing. In a preclinical study, we demonstrated that erythromycin reduces the toxicity of abnormal RNA and ameliorates the aberrant splicing and motor phenotype in DM1 model mice. Methods: This multicentre, randomised, double-blind, placebo-controlled, phase 2 trial was conducted at three centres in Japan to translate preclinical findings into practical applications in patients with DM1 by evaluating the safety and efficacy of erythromycin. Between Nov 29, 2019, and Jan 20, 2022, a total of 30 adult patients with DM1 were enrolled and randomly assigned in a 1:2:2 ratio to receive either placebo or erythromycin at two daily doses (500 mg or 800 mg) for 24 weeks. The primary outcome included the safety and tolerability of erythromycin. The secondary efficacy measures included splicing biomarkers, 6-min walk test results, muscle strength, and serum creatinine kinase (CK) values. This trial is registered with the Japan Registry of Clinical Trials, jRCT2051190069. Findings: Treatment-related gastrointestinal symptoms occurred more frequently in the erythromycin group, but all adverse events were mild to moderate and resolved spontaneously. No serious safety concerns were identified. The CK levels from baseline to week 24 decreased in the overall erythromycin group compared with the placebo group (mean change of -6.4 U/L [SD 149] vs +182.8 [SD 228]), although this difference was not statistically significant (p = 0.070). Statistically significant improvements in the overall erythromycin treated groups compared to placebo were seen for two of the eleven splicing biomarkers that were each evaluated in half of the trial sample. These were MBNL1 (p = 0.048) and CACNA1S (p = 0.042). Interpretation: Erythromycin demonstrated favourable safety and tolerability profiles in patients with DM1. A well-powered phase 3 trial is needed to evaluate efficacy, building on the preliminary findings from this study. Funding: Japan Agency for Medical Research and Development.

Comparison of Scheimpflug and Anterior Segment Optical Coherence Tomography Imaging Parameters for Japanese Patients With Fuchs Endothelial Corneal Dystrophy With and Without TCF4 Repeat Expansions.

PURPOSE: The aim of this study was to investigate the association between cytosine-thymine-guanine trinucleotide repeat (TNR) expansion in TCF4 and the clinical phenotypes of corneal densitometry or anterior segment morphology in Fuchs endothelial corneal dystrophy. METHODS: This retrospective cross-sectional study included 150 eyes from 75 Japanese consecutive patients with Fuchs endothelial corneal dystrophy. Cytosine-thymine-guanine repeat expansion of leukocyte-derived genomic DNA was analyzed through fragment analysis using polymerase chain reaction and triplet repeat primed polymerase chain reaction. Scheimpflug-based densitometry and anterior segment optical coherence tomography were applied. Corneal densitometry, and corneal and anterior segment morphology parameters were compared between patients with and without TNR expansion of 50 or more (expansion and nonexpansion groups, respectively) using a mixed model. RESULTS: The average age of the patients was 66.8 ± 13.0 years, and the modified Krachmer grading scale was 1, 2, 3, 4, 5, and 6 for 7, 32, 28, 51, 6, and 18 eyes, respectively. Sixteen patients (21%) exhibited ≥50 TNR expansion. No significant differences in sex, age, history of keratoplasty, modified Krachmer grade, and corneal densitometry in either diameter or depth were observed between the 2 groups. No significant differences in anterior segment morphology, including the anterior chamber depth and anterior chamber angle width parameters, were observed using a univariate mixed model, except for central corneal thickness (P = 0.047). However, according to the multivariate mixed model, repeat expansion was not significantly associated with central corneal thickness (P = 0.27). CONCLUSIONS: No significant differences in clinical phenotypes were found between Japanese patients having Fuchs endothelial corneal dystrophy with and without TNR expansion.

Analysis of splicing abnormalities in the white matter of myotonic dystrophy type 1 brain using RNA sequencing.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by the genomic expansion of CTG repeats, in which RNA-binding proteins, such as muscleblind-like protein, are sequestered in the nucleus, and abnormal splicing is observed in various genes. Although abnormal splicing occurs in the brains of patients with DM1, its relation to central nervous system symptoms is unknown. Several imaging studies have indicated substantial white matter defects in patients with DM1. Here, we performed RNA sequencing and analysis of CTG repeat lengths in the frontal lobe of patients with DM1, separating the gray matter and white matter, to investigate splicing abnormalities in the DM1 brain, especially in the white matter. Several genes showed similar levels of splicing abnormalities in both gray and white matter, with an observable trend toward an increased number of repeats in the gray matter. These findings suggest that white matter defects in DM1 stem from aberrant RNA splicing in both gray and white matter. Notably, several of the genes displaying abnormal splicing are recognized as being dominantly expressed in astrocytes and oligodendrocytes, leading us to hypothesize that splicing defects in the white matter may be attributed to abnormal RNA splicing in glial cells.

Contribution of Complement, Microangiopathy and Inflammation in Idiopathic Inflammatory Myopathies.

PURPOSE OF REVIEW: Idiopathic inflammatory myopathies (IIMs) are a heterogeneous group characterized by muscle weakness and skin symptoms and are categorized into six subtypes: dermatomyositis (DM), polymyositis (PM), anti-synthetase syndrome (ASS), immune-mediated myopathy (IMNM), inclusion body myopathy (IBM), and overlap myositis. Myositis-specific autoantibodies were detected for the diagnosis and classification of IIM. This review highlights the pathogenic contributions of the complement system, microangiopathy, and inflammation in IIM. RECENT FINDINGS: Deposition of complement around capillaries and/or the sarcolemma was observed in muscle biopsy specimens from patients with DM, ASS, and IMNM, suggesting the pathomechanism of complement-dependent muscle and endothelial cell injury. A recent study using human muscle microvascular endothelial cells showed that Jo-1 antibodies from ASS induce complement-dependent cellular cytotoxicity in vitro. Based on both clinical and pathological observations, antibody- and complement-mediated microangiopathy may contribute to the development of DM and anti-Jo-1 ASS. Juvenile DM is characterized by the loss of capillaries, perivascular inflammation, and small-vessel angiopathies, which may be related to microinfarction and perifascicular atrophy. Several serum biomarkers that reflect the IFN1 signature and microangiopathy are elevated in patients with DM. The pathological observation of myxovirus resistance protein A (MxA), which suggests a type 1 interferon (IFN1) signature in DM, supports the diagnosis and further understanding of the pathomechanism of IIM. A recent report showed that an increase in triggering receptor expressed on myeloid cells (TREM-1) around perimysial blood vessels and muscles in patients with IIM plays a role in triggering inflammation and promoting the migration of inflammatory cells by secreting proinflammatory cytokines, such as tumor necrosis factor α. SUMMARY: The deposition of complement in muscles and capillaries is a characteristic feature of DM, ASS, and IMNM. Microangiopathy plays a pathogenic role in DM, possibly resulting in perifascicular atrophy. Further understanding of the detailed pathomechanism regarding complement, microangiopathy, and inflammation may lead to novel therapeutic approaches for IIM.

Intracerebral Distribution of CAG Repeat-Binding Small Molecule Visualized by Whole-Brain Imaging.

Understanding the pharmacokinetics of drug candidates of interest in the brain and evaluating drug delivery to the brain are important for developing drugs targeting the brain. Previously, we demonstrated that a CAG repeat-binding small molecule, naphthyridine-azaquinolone (NA), resulted in repeat contraction in mouse models of dentatorubral-pallidoluysian atrophy and Huntington's disease caused by aberrant expansion of CAG repeats. However, the intracerebral distribution and drug deliverability of NA remain unclear. Here, we report three-dimensional whole-brain imaging of an externally administered small molecule using tissue clearing and light sheet fluorescence microscopy (LSFM). We designed and synthesized an Alexa594-labeled NA derivative with a primary amine for whole-brain imaging (NA-Alexa594-NH2), revealing the intracerebral distribution of NA-Alexa594-NH2 after intraparenchymal and intracerebroventricular administrations by whole-brain imaging combined with tissue clearing and LSFM. We also clarified that intranasally administered NA-Alexa594-NH2 was delivered into the brain via multiple nose-to-brain pathways by tracking the time-dependent change in the intracerebral distribution. Whole-brain imaging of small molecules by tissue clearing and LSFM is useful for elucidating not only the intracerebral distribution but also the drug delivery pathways into the brain.

Antineutrophil cytoplasmic antibody-associated vasculitis with predominant truncal muscle weakness: a retrospective case series.

Introduction: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) frequently leads to mononeuritis multiplex, which are characterized by distal weakness associated with sensory disturbances. Although AAV has also been reported to be associated with myopathy, the pathogenesis and characteristics remain unclear. We aimed to show the clinical and laboratory findings in AAV-associated myopathy. Methods: This retrospective single-center study included patients with the diagnosis of AAV who had been admitted to the neurology department and had biopsy specimens of muscle and/or nerve tissue. Results: We identified four patients with a distinct clinical presentation of muscle weakness in the trunk and proximal limbs. The weakness resembled that of inflammatory muscle disease. These patients denied symptoms associated with neuropathy, and had normal serum creatine kinase (CK) levels. Needle electromyography (needle EMG) showed spontaneous electrical activity at rest, and results of magnetic resonance imaging (MRI) suggested inflammatory myopathy. Muscle biopsy specimens from all four patients revealed vasculitis and inflammation in proximity to the affected vessels, without any discernible characteristics of other myopathies. The patients also complained of symptoms affecting other organs, such as the ears and kidneys, which is typical of AAV cases. Remission induction therapy, such as cyclophosphamide pulse therapy in addition to oral prednisolone, were effective for all four patients. However, relapses occurred in two patients during maintenance therapy and two patients died of aspiration pneumonia. Discussion: The clinical course of our patients might represent a subtype of AAV that is characterized by muscle weakness of the trunk and proximal extremities and arises from vasculitis within the muscles. The clinical manifestations of our patients were similar to those of patients with inflammatory myopathy with regard to the distribution of muscle weakness, MRI and needle EMG findings. However, there are notable differences between AAV associated myopathy vs. inflammatory myositis like dermatomyositis; (1) the absence of elevated CK levels, (2) the presence of complications in other organs, (3) distinct pathological findings, and (4) severe outcomes. Awareness that AAV patients with muscle involvement could have a subtype of AAV that seriously affects various organs is critical for an accurate diagnosis and effective therapeutic management.

Antagonistic roles of canonical and Alternative-RPA in disease-associated tandem CAG repeat instability.

Expansions of repeat DNA tracts cause >70 diseases, and ongoing expansions in brains exacerbate disease. During expansion mutations, single-stranded DNAs (ssDNAs) form slipped-DNAs. We find the ssDNA-binding complexes canonical replication protein A (RPA1, RPA2, and RPA3) and Alternative-RPA (RPA1, RPA3, and primate-specific RPA4) are upregulated in Huntington disease and spinocerebellar ataxia type 1 (SCA1) patient brains. Protein interactomes of RPA and Alt-RPA reveal unique and shared partners, including modifiers of CAG instability and disease presentation. RPA enhances in vitro melting, FAN1 excision, and repair of slipped-CAGs and protects against CAG expansions in human cells. RPA overexpression in SCA1 mouse brains ablates expansions, coincident with decreased ATXN1 aggregation, reduced brain DNA damage, improved neuron morphology, and rescued motor phenotypes. In contrast, Alt-RPA inhibits melting, FAN1 excision, and repair of slipped-CAGs and promotes CAG expansions. These findings suggest a functional interplay between the two RPAs where Alt-RPA may antagonistically offset RPA's suppression of disease-associated repeat expansions, which may extend to other DNA processes.

Efficacy confirmation study of aceneuramic acid administration for GNE myopathy in Japan.

BACKGROUND: A rare muscle disease, GNE myopathy is caused by mutations in the GNE gene involved in sialic acid biosynthesis. Our recent phase II/III study has indicated that oral administration of aceneuramic acid to patients slows disease progression. METHODS: We conducted a phase III, randomized, placebo-controlled, double-blind, parallel-group, multicenter study. Participants were assigned to receive an extended-release formulation of aceneuramic acid (SA-ER) or placebo. Changes in muscle strength and function over 48 weeks were compared between treatment groups using change in the upper extremity composite (UEC) score from baseline to Week 48 as the primary endpoint and the investigator-assessed efficacy rate as the key secondary endpoint. For safety, adverse events, vital signs, body weight, electrocardiogram, and clinical laboratory results were monitored. RESULTS: A total of 14 patients were enrolled and given SA-ER (n = 10) or placebo (n = 4) tablets orally. Decrease in least square mean (LSM) change in UEC score at Week 48 with SA-ER (- 0.115 kg) was numerically smaller as compared with placebo (- 2.625 kg), with LSM difference (95% confidence interval) of 2.510 (- 1.720 to 6.740) kg. In addition, efficacy was higher with SA-ER as compared with placebo. No clinically significant adverse events or other safety concerns were observed. CONCLUSIONS: The present study reproducibly showed a trend towards slowing of loss of muscle strength and function with orally administered SA-ER, indicating supplementation with sialic acid might be a promising replacement therapy for GNE myopathy. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (NCT04671472).

Phase II/III Study of Aceneuramic Acid Administration for GNE Myopathy in Japan.

BACKGROUND: GNE myopathy is an ultra-rare muscle disease characterized by a reduction in the synthesis of sialic acid derived from pathogenic variants in the GNE gene. No treatment has been established so far. OBJECTIVE: We evaluated the safety and efficacy of oral supplementation of aceneuramic acid in patients with GNE myopathy. METHODS: This multicenter, placebo-controlled, double-blind study comprised genetically confirmed GNE myopathy patients in Japan who were randomly assigned into treatment groups of sialic acid-extended release (SA-ER) tablets (6 g/day for 48 weeks) or placebo groups (4:1). The primary endpoint of effectiveness was set as the change in total upper limb muscle strength (upper extremity composite [UEC] score) from the start of administration to the final evaluation time point. RESULTS: Among the 20 enrolled patients (SA-ER group, 16; placebo group, 4), 19 completed this 48-week study. The mean value of change in UEC score (95% confidence interval [CI]) at 48 weeks was -0.1 kg (-2.1 to 2.0) in the SA-ER group and -5.1 kg (-10.4 to 0.3) in the placebo group. The least squares mean difference (95% CI) between the groups in the covariance analysis was 4.8 kg (-0.3 to 9.9; P = 0.0635). The change in UEC score at 48 weeks was significantly higher in the SA-ER group compared with the placebo group (P = 0.0013) in the generalized estimating equation test repeated measurement analysis. In one patient in the SA-ER group, who was found to be pregnant 2 weeks after drug administration fetal death with tangled umbilical cord occurred at 13 weeks after the discontinuation of treatment. No other serious adverse effects were observed. CONCLUSIONS: The present study indicates that oral administration of SA-ER tablets is effective and safe in patients with GNE myopathy in Japan.

Quercetin selectively reduces expanded repeat RNA levels in models of myotonic dystrophy.

Myotonic dystrophy is a multisystemic neuromuscular disease caused by either a CTG repeat expansion in DMPK (DM1) or a CCTG repeat expansion in CNBP (DM2). Transcription of the expanded alleles produces toxic gain-of-function RNA that sequester the MBNL family of alternative splicing regulators into ribonuclear foci, leading to pathogenic mis-splicing. There are currently no approved treatments that target the root cause of disease which is the production of the toxic expansion RNA molecules. In this study, using our previously established HeLa DM1 repeat selective screening platform, we identified the natural product quercetin as a selective modulator of toxic RNA levels. Quercetin treatment selectively reduced toxic RNA levels and rescued MBNL dependent mis-splicing in DM1 and DM2 patient derived cell lines and in the HSALR transgenic DM1 mouse model where rescue of myotonia was also observed. Based on our data and its safety profile for use in humans, we have identified quercetin as a priority disease-targeting therapeutic lead for clinical evaluation for the treatment of DM1 and DM2.

Deregulations of miR-1 and its target Multiplexin promote dilated cardiomyopathy associated with myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults. It is caused by the excessive expansion of noncoding CTG repeats, which when transcribed affects the functions of RNA-binding factors with adverse effects on alternative splicing, processing, and stability of a large set of muscular and cardiac transcripts. Among these effects, inefficient processing and down-regulation of muscle- and heart-specific miRNA, miR-1, have been reported in DM1 patients, but the impact of reduced miR-1 on DM1 pathogenesis has been unknown. Here, we use Drosophila DM1 models to explore the role of miR-1 in cardiac dysfunction in DM1. We show that miR-1 down-regulation in the heart leads to dilated cardiomyopathy (DCM), a DM1-associated phenotype. We combined in silico screening for miR-1 targets with transcriptional profiling of DM1 cardiac cells to identify miR-1 target genes with potential roles in DCM. We identify Multiplexin (Mp) as a new cardiac miR-1 target involved in DM1. Mp encodes a collagen protein involved in cardiac tube formation in Drosophila. Mp and its human ortholog Col15A1 are both highly enriched in cardiac cells of DCM-developing DM1 flies and in heart samples from DM1 patients with DCM, respectively. When overexpressed in the heart, Mp induces DCM, whereas its attenuation rescues the DCM phenotype of aged DM1 flies. Reduced levels of miR-1 and consecutive up-regulation of its target Mp/Col15A1 might be critical in DM1-associated DCM.

Pharmacotherapy alleviates pathological changes in human direct reprogrammed neuronal cell model of myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is a trinucleotide repeat disorder affecting multiple organs. However, most of the research is focused on studying and treating its muscular symptoms. On the other hand, despite the significant impact of the neurological symptoms on patients' quality of life, no drug therapy was studied due to insufficient reproducibility in DM1 brain-specific animal models. To establish DM1 neuronal model, human skin fibroblasts were directly converted into neurons by using lentivirus expressing small hairpin RNA (shRNA) against poly-pyrimidine tract binding protein (PTBP). We found faster degeneration in DM1 human induced neurons (DM1 hiNeurons) compared to control human induced neurons (ctrl hiNeurons), represented by lower viability from 10 days post viral-infection (DPI) and abnormal axonal growth at 15 DPI. Nuclear RNA foci were present in most of DM1 hiNeurons at 10 DPI. Furthermore, DM1 hiNeurons modelled aberrant splicing of MBNL1 and 2, MAPT, CSNK1D and MPRIP at 10 DPI. We tested two drugs that were shown to be effective for DM1 in non-neuronal model and found that treatment of DM1 hiNeurons with 100 nM or 200 nM actinomycin D (ACT) for 24 h resulted in more than 50% reduction in the number of RNA foci per nucleus in a dose dependent manner, with 16.5% reduction in the number of nuclei containing RNA foci at 200 nM and treatment with erythromycin at 35 µM or 65 µM for 48 h rescued mis-splicing of MBNL1 exon 5 and MBNL 2 exons 5 and 8 up to 17.5%, 10% and 8.5%, respectively. Moreover, erythromycin rescued the aberrant splicing of MAPT exon 2, CSNK1D exon 9 and MPRIP exon 9 to a maximum of 46.4%, 30.7% and 19.9%, respectively. These results prove that our model is a promising tool for detailed pathogenetic examination and novel drug screening for the nervous system.

Cell type-specific abnormalities of central nervous system in myotonic dystrophy type 1.

Myotonic dystrophy type 1 is a multisystem genetic disorder involving the muscle, heart and CNS. It is caused by toxic RNA transcription from expanded CTG repeats in the 3'-untranslated region of DMPK, leading to dysregulated splicing of various genes and multisystemic symptoms. Although aberrant splicing of several genes has been identified as the cause of some muscular symptoms, the pathogenesis of CNS symptoms prevalent in patients with myotonic dystrophy type 1 remains unelucidated, possibly due to a limitation in studying a diverse mixture of different cell types, including neuronal cells and glial cells. Previous studies revealed neuronal loss in the cortex, myelin loss in the white matter and the presence of axonal neuropathy in patients with myotonic dystrophy type 1. To elucidate the CNS pathogenesis, we investigated cell type-specific abnormalities in cortical neurons, white matter glial cells and spinal motor neurons via laser-capture microdissection. We observed that the CTG repeat instability and cytosine-phosphate-guanine (CpG) methylation status varied among the CNS cell lineages; cortical neurons had more unstable and longer repeats with higher CpG methylation than white matter glial cells, and spinal motor neurons had more stable repeats with lower methylation status. We also identified splicing abnormalities in each CNS cell lineage, such as DLGAP1 in white matter glial cells and CAMKK2 in spinal motor neurons. Furthermore, we demonstrated that aberrant splicing of CAMKK2 is associated with abnormal neurite morphology in myotonic dystrophy type 1 motor neurons. Our laser-capture microdissection-based study revealed cell type-dependent genetic, epigenetic and splicing abnormalities in myotonic dystrophy type 1 CNS, indicating the significant potential of cell type-specific analysis in elucidating the CNS pathogenesis.

Expanded CUG Repeat RNA Induces Premature Senescence in Myotonic Dystrophy Model Cells.

Myotonic dystrophy type 1 (DM1) is a dominantly inherited disorder due to a toxic gain of function of RNA transcripts containing expanded CUG repeats (CUGexp). Patients with DM1 present with multisystemic symptoms, such as muscle wasting, cognitive impairment, cataract, frontal baldness, and endocrine defects, which resemble accelerated aging. Although the involvement of cellular senescence, a critical component of aging, was suggested in studies of DM1 patient-derived cells, the detailed mechanism of cellular senescence caused by CUGexp RNA remains unelucidated. Here, we developed a DM1 cell model that conditionally expressed CUGexp RNA in human primary cells so that we could perform a detailed assessment that eliminated the variability in primary cells from different origins. Our DM1 model cells demonstrated that CUGexp RNA expression induced cellular senescence by a telomere-independent mechanism. Furthermore, the toxic RNA expression caused mitochondrial dysfunction, excessive reactive oxygen species production, and DNA damage and response, resulting in the senescence-associated increase of cell cycle inhibitors p21 and p16 and secreted mediators insulin-like growth factor binding protein 3 (IGFBP3) and plasminogen activator inhibitor-1 (PAI-1). This study provides unequivocal evidence of the induction of premature senescence by CUGexp RNA in our DM1 model cells.

Cellular Senescence and Aging in Myotonic Dystrophy.

Myotonic dystrophy (DM) is a dominantly inherited multisystemic disorder affecting various organs, such as skeletal muscle, heart, the nervous system, and the eye. Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are caused by expanded CTG and CCTG repeats, respectively. In both forms, the mutant transcripts containing expanded repeats aggregate as nuclear foci and sequester several RNA-binding proteins, resulting in alternative splicing dysregulation. Although certain alternative splicing events are linked to the clinical DM phenotypes, the molecular mechanisms underlying multiple DM symptoms remain unclear. Interestingly, multi-systemic DM manifestations, including muscle weakness, cognitive impairment, cataract, and frontal baldness, resemble premature aging. Furthermore, cellular senescence, a critical contributor to aging, is suggested to play a key role in DM cellular pathophysiology. In particular, several senescence inducers including telomere shortening, mitochondrial dysfunction, and oxidative stress and senescence biomarkers such as cell cycle inhibitors, senescence-associated secretory phenotype, chromatin reorganization, and microRNA have been implicated in DM pathogenesis. In this review, we focus on the clinical similarities between DM and aging, and summarize the involvement of cellular senescence in DM and the potential application of anti-aging DM therapies.

CAG repeat-binding small molecule improves motor coordination impairment in a mouse model of Dentatorubral-pallidoluysian atrophy.

Dentatorubral-pallidoluysian atrophy (DRPLA) is a devastating genetic disease presenting myoclonus, epilepsy, ataxia, and dementia. DRPLA is caused by the expansion of a CAG repeat in the ATN1 gene. Aggregation of the polyglutamine-expanded ATN1 protein causes neuro-degeneration of the dentatorubral and pallidoluysian systems. The expanded CAG repeats are unstable, and ongoing repeat expansions contribute to disease onset, progression, and severity. Inducing contractions of expanded repeats can be a means to treat DRPLA, for which no disease-modifying or curative therapies exist at present. Previously, we characterized a small molecule, naphthyridine-azaquinolone (NA), which binds to CAG slip-out structures and induces repeat contraction in Huntington's disease mice. Here, we demonstrate that long-term intracerebroventricular infusion of NA leads to repeat contraction, reductions in mutant ATN1 aggregation, and improved motor phenotype in a murine model of DRPLA. Furthermore, NA-induced contraction resulted in the modification of repeat-length-dependent dysregulation of gene expression profiles in DRPLA mice. Our study reveals the therapeutic potential of repeat contracting small molecules for repeat expansion disorders, such as DRPLA.

FAN1 exo- not endo-nuclease pausing on disease-associated slipped-DNA repeats: A mechanism of repeat instability.

Ongoing inchworm-like CAG and CGG repeat expansions in brains, arising by aberrant processing of slipped DNAs, may drive Huntington's disease, fragile X syndrome, and autism. FAN1 nuclease modifies hyper-expansion rates by unknown means. We show that FAN1, through iterative cycles, binds, dimerizes, and cleaves slipped DNAs, yielding striking exo-nuclease pauses along slip-outs: 5'-C↓A↓GC↓A↓G-3' and 5'-C↓T↓G↓C↓T↓G-3'. CAG excision is slower than CTG and requires intra-strand A·A and T·T mismatches. Fully paired hairpins arrested excision, whereas disease-delaying CAA interruptions further slowed excision. Endo-nucleolytic cleavage is insensitive to slip-outs. Rare FAN1 variants are found in individuals with autism with CGG/CCG expansions, and CGG/CCG slip-outs show exo-nuclease pauses. The slip-out-specific ligand, naphthyridine-azaquinolone, which induces contractions of expanded repeats in vivo, requires FAN1 for its effect, and protects slip-outs from FAN1 exo-, but not endo-, nucleolytic digestion. FAN1's inchworm pausing of slip-out excision rates is well suited to modify inchworm expansion rates, which modify disease onset and progression.

Alpha-synuclein dynamics in induced pluripotent stem cell-derived dopaminergic neurons from a Parkinson's disease patient (PARK4) with SNCA triplication.

Parkinson's disease (PD) is a neurodegenerative disorder caused by the selective loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). Lewy bodies (LBs), another histological hallmark of PD, are observed in patients with familial or sporadic PD. The therapeutic potential of reducing the accumulation of α-synuclein, a major LB component, has been investigated, but it remains unknown whether the formation of LBs results in the loss of DA neurons. PARK4 patients exhibit multiplication of the α-synuclein gene (SNCA) without any pathological mutations, but their symptoms develop relatively early. Therefore, study of PARK4 might help elucidate the mechanism of α-synuclein aggregation. In this study, we investigated the dynamics of α-synuclein during the early stage of immature DA neurons, which were differentiated from human-induced pluripotent stem cells (hiPSCs) derived from either a PARK4 patient with SNCA triplication or a healthy donor. We observed increased α-synuclein accumulation in PARK4 hiPSC-derived DA neurons relative to those derived from healthy donor hiPSCs. Interestingly, α-synuclein accumulation disappeared over time in the PARK4 patient-derived DA neurons. Moreover, an SNCA-specific antisense oligonucleotide could reduce α-synuclein levels during the accumulation stage. These observations may help reveal the mechanisms that regulate α-synuclein levels, which may consequently be useful in the development of new therapies for patients with sporadic or familial PD.

Targeting Expanded Repeats by Small Molecules in Repeat Expansion Disorders.

Recent technological advancements in genetic analysis have allowed for the consecutive discovery and elucidation of repeat expansion disorders: diseases caused by the abnormal expansion of repeat sequences in the genome. Many of these repeat expansion disorders are neurodegenerative movement disorders. Radical cures for these disorders have yet to be established. Although conventional treatments for repeat expansion disorders have mainly targeted the abnormal mRNA and proteins encoded by the affected genes, therapeutic approaches targeting repeat DNA, the root cause of repeat disorders, is also being explored in current research. In particular, a small molecule has been found that binds to abnormally expanded CAG repeats, the cause of Huntington's disease, and shortens them. Such small molecules targeting nucleic acids are expected to be developed into groundbreaking therapeutic drugs capable of ameliorating the symptoms of repeat expansion disorders and preventing their onset. © 2020 International Parkinson and Movement Disorder Society.

Alternative splicing of clathrin heavy chain contributes to the switch from coated pits to plaques.

Clathrin function directly derives from its coat structure, and while endocytosis is mediated by clathrin-coated pits, large plaques contribute to cell adhesion. Here, we show that the alternative splicing of a single exon of the clathrin heavy chain gene (CLTC exon 31) helps determine the clathrin coat organization. Direct genetic control was demonstrated by forced CLTC exon 31 skipping in muscle cells that reverses the plasma membrane content from clathrin plaques to pits and by promoting exon inclusion that stimulated flat plaque assembly. Interestingly, mis-splicing of CLTC exon 31 found in the severe congenital form of myotonic dystrophy was associated with reduced plaques in patient myotubes. Moreover, forced exclusion of this exon in WT mice muscle induced structural disorganization and reduced force, highlighting the contribution of this splicing event for the maintenance of tissue homeostasis. This genetic control on clathrin assembly should influence the way we consider how plasticity in clathrin-coated structures is involved in muscle development and maintenance.